Crystal Structure of a GH3 ß-Glucosidase from the Thermophilic Fungus Chaetomium thermophilum.

Beta-glucosidases (ß-glucosidases) have attracted considerable attention in recent years for use in various biotechnological applications. They are also essential enzymes for lignocellulose degradation in biofuel production. However, cost-effective biomass conversion requires the use of highly efficient enzymes. Thus, the search for new enzymes as better alternatives of the currently available enzyme preparations is highly important. Thermophilic fungi are nowadays considered as a promising source of enzymes with improved stability. Here, the crystal structure of a family GH3 ß-glucosidase from the thermophilic fungus Chaetomium thermophilum (CtBGL) was determined at a resolution of 2.99 Å. The structure showed the three-domain architecture found in other ß-glucosidases with variations in loops and linker regions. The active site catalytic residues in CtBGL were identified as Asp287 (nucleophile) and Glu517 (acid/base). Structural comparison of CtBGL with Protein Data Bank (PDB)-deposited structures revealed variations among glycosylated Asn residues. The enzyme displayed moderate glycosylation compared to other GH3 family ß-glucosidases with similar structure. A new glycosylation site at position Asn504 was identified in CtBGL. Moreover, comparison with respect to several thermostability parameters suggested that glycosylation and charged residues involved in electrostatic interactions may contribute to the stability of the enzyme at elevated temperatures. The reported CtBGL structure provides additional insights into the family GH3 enzymes and could offer new ideas for further improvements in ß-glucosidases for more efficient use in biotechnological applications regarding cellulose degradation.

Crystal structure and biochemical characterization of a manganese superoxide dismutase from Chaetomium thermophilum.

A manganese superoxide dismutase from the thermophilic fungus Chaetomium thermophilum (CtMnSOD) was expressed in Pichia pastoris and purified to homogeneity. Its optimal temperature was 60°C with approximately 75% of its activity retained after incubation at 70°C for 60min. Recombinant yeast cells carrying C. thermophilum mnsod gene exhibited higher stress resistance to salt and oxidative stress-inducing agents than control yeast cells. In an effort to provide structural insights, CtMnSOD was crystallized and its structure was determined at 2.0Å resolution. The overall architecture of CtMnSOD was found similar to other MnSODs with highest structural similarities obtained against a MnSOD from the thermotolerant fungus Aspergillus fumigatus. In order to explain its thermostability, structural and sequence analysis of CtMnSOD with other MnSODs was carried out. An increased number of charged residues and an increase in the number of intersubunit salt bridges and the Thr:Ser ratio were identified as potential reasons for the thermostability of CtMnSOD.

Directed evolution and structural prediction of cellobiohydrolase II from the thermophilic fungus Chaetomium thermophilum.

Cellulases can be engineered with enhanced properties for broad use in scientific and industrial applications. In this study, the wild-type cbh2 gene of the thermophilic fungus Chaetomium thermophilum encoding cellobiohydrolase II (CBHII) was mutagenized through in vitro directed evolution. The resulting Pichia pastoris yeast library was screened, and two transformants were selected for enhanced CBHII activities that were not attributed to increased gene copy numbers. The optimum fermentation times of the two mutant transformants were shortened to 4-5 days after methanol induction compared to 6 days for the wild-type. The optimum reaction temperature (60 °C) and pH level (5 or 6) of the mutant CBHII proteins, designated CBHIIX16 and CBHIIX305, were higher than those of wild-type CBHII (50 °C and pH 4). Kept at 80 °C for 1 h, CBHIIX16 and CBHIIX305 retained >50% of their activities, while the wild-type CBHII lost all activity. Sequence analysis of CBHIIX16 and CBHIIX305 revealed that they contained five and six mutated amino acids, respectively. Structural modeling confirmed the presence of carbohydrate binding type-1 and catalytic domains, where the hydrogen bond numbers between the 227th and 203rd amino acids were increased, which perhaps contributed to the elevated enzyme stability. Therefore, the two CBHII mutants selected for increased enzymatic activities also demonstrated elevated optimum reaction temperature and pH levels and enhanced thermal stability. These properties may be beneficial in practical applications for CBHII.

Expression of a novel thermostable Cu, Zn-superoxide dismutase from Chaetomium thermophilum in Pichia pastoris and its antioxidant properties.

A new superoxide dismutase (SOD) gene from the thermophilic fungus Chaetomium thermophilum (Ctsod) was cloned and expressed in Pichia pastoris and its gene product was characterized. The specific activity of the purified CtSOD was 2,170 U/mg protein. The enzyme was inactivated by KCN and H(2)O(2) but not by NaN(3), confirming that it belonged to the type of Cu, ZnSOD. The amino acid residues involved in coordinating copper and zinc were conserved. The recombinant CtSOD exhibited optimum activity at pH 6.5 and 60°C. The enzyme retained 65% of the maximum activity at 70°C for 60 min and the half-life was 22 and 7 min at 80 and 90°C, respectively. The recombinant yeast exhibited higher stress resistance than the control yeast cells to salt and superoxide-generating agents, such as paraquat and menadione.

Crystal Structure of a Cu,Zn Superoxide Dismutase From the Thermophilic Fungus Chaetomium thermophilum.

BACKGROUND: Thermophilic fungi have recently emerged as a promising source of thermostable enzymes. Superoxide dismutases are key antioxidant metalloenzymes with promising therapeutic effects in various diseases, both acute and chronic. However, structural heterogeneity and low thermostability limit their therapeutic efficacy. OBJECTIVE: Although several studies from hypethermophilic superoxide dismutases (SODs) have been reported, information about Cu,Zn-SODs from thermophilic fungi is scarce. Chaetomium thermophilum is a thermophilic fungus that could provide proteins with thermophilic properties. METHODS: The enzyme was expressed in Pichia pastoris cells and crystallized using the vapor-diffusion method. X-ray data were collected, and the structure was determined and refined to 1.56 Å resolution. Structural analysis and comparisons were carried out. RESULTS: The presence of 8 molecules (A through H) in the asymmetric unit resulted in four different interfaces. Molecules A and F form the typical homodimer which is also found in other Cu,Zn- SODs. Zinc was present in all subunits of the structure while copper was found in only four subunits with reduced occupancy (C, D, E and F). CONCLUSION: The ability of the enzyme to form oligomers and the elevated Thr:Ser ratio may be contributing factors to its thermal stability. Two hydrophobic residues that participate in interface formation and are not present in other CuZn-SODs may play a role in the formation of new interfaces and the oligomerization process. The CtSOD crystal structure reported here is the first Cu,Zn-SOD structure from a thermophilic fungus.

Expression, purification and crystallization of Chaetomium thermophilum Cu,Zn superoxide dismutase.

Cu,Zn superoxide dismutase (Cu,ZnSOD) from the thermophilic fungus Chaetomium thermophilum was expressed in Pichia pastoris and purified. Crystals were grown in over 120 conditions but only those produced with 1.4 M sodium potassium phosphate pH 8.2 as precipitant were suitable for structural studies. Data were collected to 1.9 A resolution at 100 K from a single crystal using a synchrotron-radiation source. The crystals belonged to space group P6(1)/P6(5), with unit-cell parameters a=90.2, c=314.5 A and eight molecules in the asymmetric unit. Elucidation of the crystal structure will provide insights into the active site of the enzyme and a better understanding of the structure-activity relationship, assembly and thermal stability of Cu,ZnSODs.

The gene fpk1, encoding a cAMP-dependent protein kinase catalytic subunit homolog, is required for hyphal growth, spore germination, and plant infection in Fusarium verticillioides.

Fusarium verticillioides is an important pathogen of maize responsible for ear rots, stalk rots and seeding blight worldwide. During the past decade F. verticillioides has caused several severe epidemics of maize seeding blight in many areas of china, which lead to significant losses. In order to understand molecular mechanisms regulating fungal development and pathogenicity in the pathogen, we isolated and characterized the gene fpk1 (GenBank accession NO. EF405959) encoding catalytic subunit of cAMP-dependent protein kinase, which include 1854 bp DNA sequence from ATG to TAA, with 1680 bp coding region, three intron (their length: 66bp, 54bp and 54bp), and the predicated protein had 559 aa. The mutantdeltafpk1, which was disrupted of fpk1 gene, showed reduced vegetative growth, fewer and shorter aerial mycelia, strongly impaired conidiation and reduced spore germination rate. After germinating, the fresh hypha was stubby and lack of branch. When inoculated in susceptible maize varieties, the infection of the mutantdeltafpk1 was delayed and the infection efficiency was reduced than that of the wild-type. All this indicated that the gene fpk1 participated in hyphal growth, conidiophore producing, spore germination and virulence in F. verticillioides.

Purification and characterization of a thermostable MnSOD from the thermophilic fungus Chaetomium thermophilum.

A thermostable superoxide dismutase (SOD) from the culture supernatant of a thermophilic fungus Chaetomium thermophilum strain CT2 was purified to homogeneity by fractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-sepharose, phenyl-sepharose hydrophobic interaction chromatography. The pure SOD had a specific activity of 115.77 U/mg of protein and was purified 7.49-fold, with a yield of 14.4%. The molecular mass of a single band of the enzyme was estimated to be 23.5 kDa, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using gel filtration on Sephacryl S-100, the molecular mass was estimated to be 94.4 kDa, indicating that this enzyme was composed of four identical subunits of 23.5 kDa each. The SOD was found to be inhibited by NaN3, but not by KCN and H2O2. Atomic absorption spectrophotometric analysis showed that the content of Mn was 2.05 microg/mg of protein and Fe was not detected in the purified enzyme. These results suggested that the SOD in C. thermophilum was the manganese superoxide dismutase type. N-terminal amino acid sequencing (10 residues) was KX (X is uncertain) TLPDLKYD. The N-terminal amino acid sequencing homologies to other MnSod also indicated that it was a manganese-containing superoxide dismutase. The SOD exhibited maximal activity at pH 7.5 and optimum temperature at 60 C. It was thermostable at 50 and 60 C and retained 60% activity after 60 min at 70 C. The half-life of the SOD at 80 C was approximately 25 min and even retained 20% activity after 30 min at 90 C.

Molecular cloning and characterization of a putative protein kinase gene from the thermophilic fungus Thermomyces lanuginosus.

Based on the conserved amino acid sequence (DLKPEN) of serine-threonine protein kinase from several fungi, a degenerate primer was designed and synthesized. Total RNA was isolated from the thermophilic fungus Thermomyces lanuginosus. Using RACE-PCR, full-length cDNA of a putative serine-threonine protein kinase gene was cloned from T. lanuginosus. The full-length cDNA of T. lanuginosus protein kinase was 2551 bp and contained an 1806 bp open reading frame encoding a putative protein kinase precursor of 601 amino acid residues. Sequencing analysis showed that the cloned cDNA of T. lanuginosus had consensus protein kinase sequences. Conservative amino acid subdomains which most serine-threonine kinases contain can be found in the deduced amino acid sequence of T. lanuginosus putative protein kinase. Comparison results showed that the deduced amino acid sequence of T. lanuginosus putative protein kinase was highly homologous to that of Neurospora crassa dis1-suppressing protein kinase Dsk1. The putative protein kinase contained three arginine/serine-rich (SR) regions and two transmembrane domains. These showed that it might be a novel putative serine-threonine protein kinase.

Purification and characterization of two thermostable proteases from the thermophilic fungus Chaetomium thermophilum.

Thermostable protease is very effective to improve the industrial processes in many fields. Two thermostable extracellular proteases from the culture supernatant of the thermophilic fungus Chaetomium thermophilum were purified to homogeneity by fractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sepharose, and PhenylSepharose hydrophobic interaction chromatography. By SDS-PAGE, the molecular mass of the two purified enzymes was estimated to be 33 kDa and 63 kDa, respectively. The two proteases were found to be inhibited by PMSF, but not by iodoacetamide and EDTA. The 33 kDa protease (PRO33) exhibited maximal activity at pH 10.0 and the 63 kDa protease (PRO63) at pH 5.0. The optimum temperature for the two proteases was 65 degrees C. The PRO33 had a K(m) value of 6.6 mM and a V(max) value of 10.31 micromol/l/min, and PRO63 17.6 mM and 9.08 micromol/l/min, with casein as substrate. They were thermostable at 60 degrees C. The protease activity of PRO33 and PRO63 remained at 67.2% and 17.31%, respectively, after incubation at 70 degrees C for 1 h. The thermal stability of the two enzymes was significantly enhanced by Ca2+. The residual activity of PRO33 and PRO63 at 70 degrees C after 60 min was approximately 88.59% and 39.2%, respectively, when kept in the buffer containing Ca2+. These properties make them applicable for many biotechnological purposes.

[cDNA cloning and expression of thermostable chitinase from thermophilic fungus Thermomyces lanuginosus].

A chitinase-encoding gene from Thermomyces lanuginosus was cloned by Rapid Amplification of cDNA Ends (RACE) using degenerated oligonucleotide primers designed from N-terminal amino acid sequence and the conserved amino acid sequence. The cloned full-length cDNA named chit, 1500bp in size, contained an OFR with 442 amino acids. The gene chit has been registered in GenBank with accession number DQ092332. The alignment results of putative amino acids sequence showed the catalytic domain of chit was high homology with the catalytic domains of the other chitinases in family-18, contained 2 conserved motifs related with catalytic activity of chitinase. The recombinant plasmid was generated by inserting the ORF of mature protein Chit into expression vector pPIC9K, and transformed into Pichia pastoris GS115. The recombinant chitinase was secreted successfully with the expression level of 0.36mg/mL and its activity could reach to 2.261U/mL after methanol induction 6d. The recombinant chitinase exhibited optimum catalytic activity at pH 5.5 and 60 degrees C. The enzyme was stable at 50 degrees C and the half life time at 65 degrees C was 40min.

[Purification and characterization of a cellobiohydrolase from the thermophilic fungus Chaetomium thermophilus CT2].

Chaetomium thermophilus CT2 was a cellulolytic fungus. It was a widely-existing saprophyte, which grower rapidly in soil. The cellulases synthesized by C. thermophilus CT2 was overall, consisting of three principal types of enzymes. The cellobiohydrolase was one of these three cellulases, which was associated with the endo-beta-1,4-glucanase and beta-glucosidase activities. C. thermophilus CT2 produced cellobiohydrolase available at 50 degrees C, when grown on ferment liquid substrate, containing 1% Avicel, 0.14% (NH4 )2SO4, 0.2% KH2PO4, 0.03% CaC2 x 2H2O, 0.03% MnSO4 x 7H2O, 0.1% peptone, 0.05% yeast extract, 0.1% Tween 80 and trace element solution at 1mL/L, containing 18mmol/L FeSO4 x 7H2O, 6.6mmol/L MnSO4, 4.8mmol/L ZnSO4 x 7H2O and 15mmol/L COCl2. A cellobiohydrolase was purified to homogeneity by an inexpensive and straightforward method for extraction of the enzyme involving fractional ammonium sulphate precipitation, ion-exchange chromatography on DEAE-Sepharose Fast Flow, gel filtration on Sephacryl S-100 and ion-exchange chromatography on Q Sepharose Fast Flow. The molecular weight of the enzyme was estimated to be 66.3kDa by 12.5% SDS-PAGE and was to be 67.1kDa by gel filtration on Sephacryl S-100 respectively. Kinetic studies of the purified cellobiohydrolase of C. thermophilus CT2 showed that the Km for p-NPC (p-trophenylbeta-d-cellobioside) was 0.956mmol/L as determined from a Lineweaver-Bark plot. Optimum enzyme activity was at 65 degrees C and pH5.0. It was thermostable at 60 degrees C and remained 20% activity after 20min at 80 degrees C. The half life time of the enzyme at 70 degrees C was 1h. It indicated that the cellobiohydrolase possessed of excellent acid stability and thermostable property. The properties of the cellobiohydrolase make it possible to be good material in scientific researches of protein thermostable mechanism and good model for designing and constructing a new type protein in industry. The enzyme may also provide instructive insight on the diversity and mechanism of cellulose degradation by C. thermophilus CT2. As a thermophilic fungus C. thermophilus CT2 is an attractive potential source of cellulases. It indicates that C. thermophilus CT2 may be a new excellent industrialized fungus for producing cellulases through molecule biology means.

Purification and characterization of a thermostable glucoamylase from Chaetomium thermophilum.

Thermostable amylolytic enzymes are currently being investigated to improve industrial processes of starch degradation. A thermostable extracellular glucoamylase (exo-1, 4-alpha-D-glucanohydrolase, E.C.3.2.1.3) from the culture supernatant of a thermophilic fungus Chaetomium thermophilum was purified to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) homogeneity by using ammonium sulfate fraction, DEAE-Sepharose Fast Flow chromatography, and Phenyl-Sepharose Fast Flow chromatography. SDS-PAGE of the purified enzyme showed a single protein band of molecular weight 64 kDa. The glucoamylase exhibited optimum catalytic activity at pH 4.0 and 65 degrees C. It was thermostable at 50 degrees C and 60 degrees C, and retained 50% activity after 60 min at 65 degrees C. The half-life of the enzyme at 70 degrees C was 20 min. N-terminal amino acid sequencing (15 residues) was AVDSYIERETPIAWN. Different metal ions showed different effects on the glucoamylase activity. Ca2+, Mg2+, Na+, and K+ enhanced the enzyme activity, whereas Fe2+, Ag+, and Hg2+ cause obvious inhibition. These properties make it applicable to other biotechnological purposes.

[Purification and properties of a thermostable chitinase from thermophilic fungus Thermomyces lanuginosus].

A thermostable extracellular chitinase from culture supernatant of a thermophilic fungus Thermomyces lanuginosus was purified to SDS-PAGE homogeneity, by using ammonium sulfate fraction, DEAE-Sepharose Fast flow chromatography, Phenyl-Sepharose Fast Flow chromatography. A molecular mass of the purified enzyme was between 48-49.8 kD determined by SDS-PAGE and gel filtration chromatography. The chitinase exhibited optimum catalytic activity at pH 4.5 and 55 degrees C respectively. It was thermostable at 50 degrees C and retained 24% activity after 20 min at 70 degrees C. The half life time of the enzyme at 65 degrees C was 25 min. Different metal ions showed different effects on the chitinase activity. Ca2+, Ba2+, Na+, K+ enhanced the enzyme activity, whereas Fe2+, Ag+, Hg2+, Cu2+ caused obvious inhibition. The Km and Vmax values of chitinase on colloidal chitin were 9.56 mg/mL and 22.12 micromol/min respectively. The chitinase showed antifungal activity aginst tested fungi to different degree.

[Induction, purification and antifungal activity of beta-1, 3-glucanase from wheat leaves].

Treatment with mercuric chloride (0.01%), salicylic acid (10.0 mg/mL) or riboflavin (1 mmol/L) induced the beta-1, 3-glucanase activity in all the three wheat varieties i.e. 331, Kangdao 680 and Lumai 23 tested, with the strongest inductive effect on variety 331 by treatment with mercuric chloride (0.01%) for 24 h. From leaves of variety 331 treated with mercuric chloride (0.01%) for 24 h, a kind of beta-1, 3-glucanase was purified by fractional precipitation with ammonium sulphate, Phenyl-Sepharose chromatography (Phenyl-Sepharose Fast Flow), ion-exchange chromatography (DEAE-Sepharose Fast Flow) and gel-filtration chromatography (Sephacryl S-100). Through SDS-PAGE and gel filtration, the molecular weight of the purified beta-1, 3-glucanase was determined to be about 52.0-53.6 kD. The purified beta-1, 3-glucanase showed antifungal activity against both Alternaria longipes and Rhizoctonia cerealis on tested plates, and inhibited the germ tube elongation and spore germination of Verticillium dahliae and Fusarium omysporum f.sp cucumerinum.

Cellulases from thermophilic fungi: recent insights and biotechnological potential.

Thermophilic fungal cellulases are promising enzymes in protein engineering efforts aimed at optimizing industrial processes, such as biomass degradation and biofuel production. The cloning and expression in recent years of new cellulase genes from thermophilic fungi have led to a better understanding of cellulose degradation in these species. Moreover, crystal structures of thermophilic fungal cellulases are now available, providing insights into their function and stability. The present paper is focused on recent progress in cloning, expression, regulation, and structure of thermophilic fungal cellulases and the current research efforts to improve their properties for better use in biotechnological applications.

The FUS3/KSS1-type MAP kinase gene FPK1 is involved in hyphal growth, conidiation and plant infection of Fusarium proliferatum.

Fusarium proliferatum is an important pathogen of maize that is responsible for ear rots, stalk rots and seeding blight worldwide. During the past decade, F. proliferatum has caused several severe epidemics of maize seedling blight in many areas of China, which led to significant losses in maize. To understand the molecular mechanisms in the fungal developmental regulation and pathogenicity, we isolated and characterized the FPK1 gene (GenBank accession No. HQ844224) encoding a MAP kinase homolog of FUS3/KSS1 in yeast. The gene includes a 1,242-bp DNA sequence from ATG to TAA, with a coding region of 1,068 bp, 3 introns (58 bp, 56 bp and 60 bp) and a predicted protein of 355 aa.The mutant ΔFPK1, which has a disruption of the FPK1 gene, showed reduced vegetative growth, fewer and shorter aerial mycelia, strongly impaired conidiation and spore germination, as well as deviant germ tube outgrowth. When the strain was inoculated in susceptible maize varieties, the infection of the mutant ΔFPK1 was delayed, and the infection efficiency was reduced compared to the wild-type strain. Complementation of the disruptions within the FPK1 open reading frame restored wild-type levels of conidiation, growth rate and virulence to maize seedlings. Our results indicated that the FPK1 gene functioned in hyphal growth, conidiation, spore germination and virulence in F. proliferatum.

Cloning, expression, and characterization of serine protease from thermophilic fungus Thermoascus aurantiacus var. levisporus.

The serine protease gene from a thermophilic fungus Thermoascus aurantiacus var. levisporus, was cloned, sequenced, and expressed in Pichia pastoris and the recombinant protein was characterized. The full-length cDNA of 2,592 bp contains an ORF of 1,482 bp encoding 494 amino acids. Sequence analysis of the deduced amino acid sequence revealed high homology with subtilisin serine proteases. The putative enzyme contained catalytic domain with active sites formed by three residues of Aspl83, His215, and Ser384. The molecular mass of the recombinant enzyme was estimated to be 59.1 kDa after overexpression in P. pastoris. The activity of recombinant protein was 115.58 U/mg. The protease exhibited its maximal activity at 50°C and pH 8.0 and kept thermostable at 60°C, and retained 60% activity after 60 min at 70° C. The protease activity was found to be inhibited by PMSF, but not by DTT or EDTA. The enzyme has broad substrate specificity such as gelatin, casein and pure milk, and exhibiting highest activity towards casein.

Two novel thermostable chitinase genes from thermophilic fungi: cloning, expression and characterization.

Two chitinase genes, Tachit1 from Thermoascus aurantiacus var. levisporus and Ctchit1 from Chaetomium thermophilum were isolated. Tachit1 and Ctchit1 encode putative single-domain proteins (TaCHIT1 and CtCHIT1) of 399 and 402 amino acid residues, respectively. The catalytic domains of TaCHIT1 and CtCHIT1 are similar to those of other fungal chitinases in family 18 of glycosyl hydrolases. TaCHIT1 and CtCHIT1 have a molecular weight of about 48.4 and 47.3kDa, respectively when produced in recombinant Pichia pastoris. The enzymes exhibited optimum catalytic activity at pH 8.0 and 50 degrees C for TaCHIT1 and at pH 5.5 and 60 degrees C for CtCHIT1. TaCHIT1 retained 95.3% of its activity after 60 min at 50 degrees C. CtCHIT1 was stable at 50 degrees C and retained 96.7% of its activity after 60 min incubation at 60 degrees C. The TaCHIT1 and CtCHIT1 produced Glc-NAc2 as the major product, when colloidal chitin was used as the substrate. The enzyme could not hydrolyze pNp-(GlcNAc), but hydrolyzed colloidal chitin, powdery chitin and chitosan. These features make these proteins potentially useful for applications requiring chitin hydrolysis at elevated temperatures.